Microtubules (Mts) are polymers of the cytoskeleton that serve to organize the cytoplasm and to act as tracks for the delivery of cargo by motor proteins. In order to perform these functions, the ends of Mts sometimes need to form stable linkages with targets, such as organelles or cortical domains. These linkages are thought to form between MT plus end-associated proteins and target-bound 'receptors.' The EB1 family of proteins is believed to be one such MT-bound protein. We propose to study the cellular functions of EB1 in Drosophila and to understand the molecular mechanisms that target it to MT plus ends by pursuing three specific aims. In the first, we will inhibit the function of EB1 in cultured cells using RNAi and use immunofluorescence- microscopy to assess the role EB1 plays in MT organization during interphase and mitosis. We will also employ real-time confocal microscopy to observe the role of inhibitory antibodies on the mitotic spindles of living fly embryos. In aim two, we will reconstitute EB1 targeting to the plus ends of MTs using in vitro assays and study the interactions between tubulin and EB1 using biochemical techniques, as well as atomic force microscopy. In the third, we will identify EB1- interacting proteins using biochemical approaches, such as affinity chromatography and immunoprecipitation, and by using two-hybrid interaction to identify EB1Q effector proteins and candidate EB1 receptors. Collectively, these studies will allow us to assign cellular functions to EB1 and to determine how this protein interacts with the MT cytoskeleton.